Immunogenic compositions to the CCK-B/gastrin receptor and methods for the treatment of tumors

ABSTRACT

The invention concerns immunogens, immunogenic compositions and method for the treatment of gastrin-dependent tumors. The immunogens comprise a peptide from the CCK-B/gastrin-receptor conjugated to a spacer and to an immunogenic carrier. The immunogens are capable of inducing antibodies in vivo which bind to the CCK-B/gastrin-receptor in tumor cells, thereby preventing growth stimulating peptide hormones from binding to the receptors, and inhibiting tumor cell growth. The immunogens also comprise antibodies against the CCK-B/gastrin-receptor for passive immunization. The invention also concerns diagnostic methods for detecting gastrin-dependent tumors in vivo or from a tissue biopsy using the antibodies of the invention.

CROSS REFERENCE TO RELATED APPLICATION

[0001] This application claims the benefit under 35 U.S.C. §119(e), ofU.S. Provisional Application No. 60/046,201 filed on May 12, 1997.

BACKGROUND OF THE INVENTION

[0002] The hormone gastrin binds to a gastrin/cholecystokinin (CCK)-Breceptor with high affinity via its 5 carboxy-terminal amino acids. TheCCK- B/gastrin receptor is a cytoplasmic membrane protein which iscoupled via a G protein to intracellular signal transduction pathwaysthat in turn control the expression of various genes.

[0003] Gastrin is a peptide hormone which occurs in two forms,tetratriacontagastrin (G34) and heptadecagastrin (G17), and issynthesized and secreted by specialized cells, G cells, that are locatedin the stomach antrum. The hormone is secreted into the circulatingblood and binds to specific cells in the stomach, namely,enterochromaffin-like (ECL) and parietal cells, that indirectly ordirectly affect stomach acid output. Historically, gastrin hormones havebeen associated with the stimulation of gastric acid secretion (Edkins,J. S. 1905). (The full citations for the references cited herein areprovided in the Reference section preceding the claims.) In recentyears, evidence has accumulated that gastrin may act as a trophic factorwithin the gastrointestinal tract (Johnson, L. 1997) and that it canpromote the growth of gastrointestinal cancers (Watson et al. 1989,Dickinson, C. J. 1995), as well as non-gastrointestinal cancersincluding small cell carcinoma of the lung (Rehfeld et al. 1989). In thepost-translational processing of gastrin, it is the “mature”carboxy-amidated form that binds to the gastrin/CCK-B receptor via thecarboxy terminus (Kopin et al. 1992).

[0004] It has been shown that several types of tumors, e.g., colorectal,stomach, pancreatic and hepatocellular adenocarcinomas possessCCK-B/gastrin receptors in their plasma membranes and that they respondto gastrin with powerful cellular proliferation (Rehfeld, J. F. 1972,Upp et al. 1989 and Watson et al. 1993). Furthermore, more recently ithas been discovered that many of these cancer cells also secrete gastrinand thus effect an autonomous proliferative pathway (Van- Solinge et al.1993, Nemeth et al. 1993 and Seva et al. 1994).

[0005] The CCK-B/gastrin receptor belongs to a family of Gprotein-coupled receptors with seven transmembrane domains with equalaffinity for both CCK and gastrin (Soll et al. 1984). This receptor wasnamed a CCK type-B receptor because it was found predominantly in thebrain (Wank et al. 1992). The receptor was subsequently found to beidentical to the peripheral CCK/gastrin receptor in the parietal and ECLcells of the stomach (Nakata et al. 1992). This receptor has been wellcharacterized in a number of normal (Fourmy et al. 1984, Grider et al.1990) and tumor tissues (Singh et al. 1990, Watson et al. 1993), andextensively studied using the rat pancreatic adenocarcinoma cell lineAR42J (Scemama et al. 1987). The AR42J CCK-B/gastrin receptor cDNA hasbeen cloned and sequenced, and it is more than 90% homologous in DNAsequence to the CCK-B/gastrin receptor in rat and human brain, and morethan 84% homologous in sequence to the canine parietal cellCCK-B/gastrin receptor cDNA (Wank, S. A. 1995), demonstrating a highsequence homology even between species.

[0006] The peptide hormones G17 and G34 bind to the CCK- B/gastrinreceptor on the cell membrane of normal cells. However, it has beenfound that G17, and not G34, stimulates the growth of gastrin-dependentcancer cells. Serum-associated G17, in particular, has the potential tostimulate the growth of colorectal tumors in an endocrine mannermediated by CCK-B/gastrin receptors (Watson et al. 1993) in the tumorcells. Gastrin-17 appears to be particularly implicated in stimulatingthe growth of colorectal adenocarcinomas due to a possible increasedaffinity for the CCK-B/gastrin receptor on the tumor cells, over othergastrin hormone species (Rehfeld 1972 and 1993). The CCK-B/gastrinreceptors were found to be expressed in a high affinity form on 56.7% ofhuman primary colorectal tumors (Upp et al. 1989). It has beenpostulated that a potential autocrine loop may also exist due toendogenous production of precursor gastrin peptides by such tumors(Van-Solinge et al. 1993 and Nemeth et al. 1993). The resulting G17ligand/receptor complex stimulates cell growth by way of secondarymessengers for regulating cell function (Ullrich et al. 1990). Thebinding of G17 to the CCK- B/gastrin receptor leads to activation ofphosphatidyl inositol breakdown, protein kinase C activation with aresultant increase in intracellular calcium ion concentration, as wellas the induction of c-fos and c-jun genes via mitogen-activated proteinkinase, which has been implicated in the regulation of cellproliferation (Tadisco et al. 1995). Additionally, gastrin binding tothe CCK-B/gastrin receptor has been associated with the subsequentincrease in phosphorylation by a tyrosine kinase, pp125FADK (focaladhesion kinase), which may also have a role in the transmission ofmitogenic signals (Tanaguchi et al. 1994).

[0007] A number of high affinity CCK-B/gastrin receptor antagonists havebeen evaluated therapeutically both in vitro and in vivo in a number ofexperimental gastrointestinal cancers. For example, proglumide, aglutamic acid derivative (Seva et al. 190; Harrison et al. 1990 andWatson et al. 1991a); Benzotript, an N-acyl derivative of tryptophan;L-365,260, a derivative of Aspercillin (Bock et al. 1989), and CI-988 amolecule that mimics the C-terminal pentapeptide sequence of CCK (Hugheset al. 1990) have been shown to effectively neutralize the effects ofexogenous gastrin on gastrointestinal tumor growth both in vitro and invivo (Watson et al. and Romani et al. 1994). However, these antagonistshave severe toxic side effects and lack specificity as they block theaction of all potential ligands of the receptor such as G34 and CCK innormal cells. Recently, highly potent and selective CCKB/gastrinreceptor antagonists such as YM022 (Yuki et al., 1997) and YF476(Takinami et al., 1997) have been also described.

[0008] Proglumide and Benzotript have been widely assessed in thepre-clinical studies. The main problem with these compounds is theirlack of potency, with relatively high concentrations required todisplace G17 (Watson et al., 1992a; Watson et al., 1992b). Despite this,proglumide and benzotript inhibited the basal and gastrin-stimulatedproliferation of a number of cell lines (Seva et al., 1990; Watson etal., 1991 a). In addition, proglumide increased the survival ofxenograft mice bearing the gastrin-sensitive mouse colon tumor, MC26 to39 days in the treated animals from 25 days in the control animals.

[0009] Due to the low specificity of this class of gastrin antagonisingagents for the gastrin/CCKB receptor, the inhibition of tumor growth maynot be effectively control with gastrin antagonists. Moreover, thecellular receptors which recognize and bind the gastrins do not bind allthe inhibitors tested (Seva et al. 1994). Thus, if complete inhibitionof gastrin binding to the receptor does not occur in the autocrinegrowth cascade, then the gastrin antagonists may be unable to block thismechanism of tumor growth promotion.

SUMMARY OF THE INVENTION

[0010] The present invention provides immunogenic compositions andimmunological methods for the treatment of gastrin-dependent tumors. Themethod comprises the active or passive immunization of a patient with ananti-CCK-B/gastrin receptor immunogen or anti-CCK-B/gastrin receptorantibodies. The antibodies produced by the immunogens are specificagainst the CCK-B/gastrin receptor on tumor cells and block thegrowth-promoting effects of gastrin on the receptors. The antibodiesprevent the peptide hormones from binding to the CCK-B/gastrin receptorson gastrin-dependent tumor cells; thus, the growth of the tumor isarrested. Furthermore, surprisingly, the antibodies specific to theNH₂-terminal end of the receptor, upon binding to the receptor, areinternalized and rapidly translocated into the cytoplasm and into thenucleus of the tumor cells. This internalization can occur as early as10 seconds after exposing the cells to the antibody. This rapidinternalization of the antibody/receptor complex in turn causes theaffected tumor cells to undergo apoptosis or suicide.

[0011] The immunogens of the invention comprise natural or syntheticpeptides derived from the human CCK-B/gastrin receptor, as theimmunomimic portion of the immunogen. The immunogens may also comprise aspacer peptide sequence attached to an end of the immunomimic peptide.The immunogen may also be conjugated to a protein carrier, such asDiphtheria toxoid, tetanus toxoid, bovine serum albumin and the like.

[0012] In one embodiment, the method of immunization against theCCK-B/gastrin receptor comprises active immunization, wherein a patientis immunized with an immunogen of the invention. The immunogenstimulates the production of antibodies against the CCK-B/gastrinreceptor on tumor cells.

[0013] The antibodies produced by the anti-CCK-B/gastrin receptorimmunogens bind to the CCK-B/gastrin receptors on tumor cells andeffectively prevent the binding of the peptide hormones to thereceptors, thereby inhibiting the autocrine growth-stimulatory pathwayof tumor cell division and ultimately the growth of the tumor.

[0014] In another embodiment of the invention, the method of treatmentcomprises passive immunization, whereby antibodies against theCCK-B/gastrin receptor are administered to a patient in a sufficientconcentration to bind to the CCK-B/gastrin receptors of the tumor cells,and the antibodies block the binding of the peptide hormones to thereceptor. The prevention of binding of the hormones to their receptorinhibits the growth-stimulus pathway of the tumor cells, therebyinhibiting the growth of the hormone-dependent tumors. In a preferredembodiment of this aspect of the invention, the antibodies for humantherapy may be chimeric, humanized, or human monoclonal antibodies whichmay be produced by methods well known in the art. In addition, theanti-CCK-B/gastrin receptor antibodies may be further conjugated tocytotoxic molecules such as cholera toxin, or to radioactive moleculeslabeled with a radionuclide, such as ¹²⁵I and ¹³¹I, to enhance thekilling of the tumor cells.

[0015] The invention also provides a method for diagnosing agastrin-responsive tumor, comprising the immunochemical detection ofgastrin-dependent (CCK-B/gastrin-containing) tumors from a tissue biopsyusing the antibodies of the invention. The specific anti-CCK-B/gastrinreceptor antibodies of the invention can be labeled with a detectionsystem utilizing compounds such as biotin, horseradish peroxidase andfluorescein to detect the CCK-B/gastrin receptors in the tumor tissueusing standard immunochemical procedures.

[0016] The invention also provides a method for diagnosing agastrin-dependent tumor, comprising the in vivo detection ofgastrin-dependent (CCK-B/gastrin receptor-containing) tumors, using theanti-CCK-B/gastrin receptor antibodies. The method comprises,administering to a patient possessing a colorectal tumor an effectivedose of radiolabeled anti-CCK-B/gastrin receptor antibodies via anintravenous injection, and imaging or detecting tumor cells havinganti-CCK-B/gastrin receptor antibodies bound to their cell membranes bystandard scintigraphic scanning procedures. In this aspect of theinvention, the anti-CCK-B/gastrin-receptor-antibodies should be labeledwith a radionuclide such as ¹¹¹Indium, ⁹⁰Yttrium, and ¹³¹I.

BRIEF DESCRIPTION OF THE DRAWINGS

[0017]FIG. 1A and 1B illustrate schematic views of the CCK-B/gastrinreceptor and its 7 transmembrane domains.

[0018]FIG. 2 shows data from ELISA assays with antibodies raised inrabbits immunized with an immunogen against Peptide 1 of theCCK-B/gastrin receptor.

[0019]FIG. 3 shows data from ELISA assays with antibodies raised inrabbits immunized with an immunogen against Peptide 4 of theCCK-B/gastrin receptor.

[0020]FIG. 4 is a graph showing data obtained from an inhibition ELISAused to assess the specificity of affinity-purified antibodies raisedagainst GRP1-DT immunogen.

[0021]FIG. 5 is a bar graph showing data on the inhibition of thebinding of ¹²⁵I-human G17 to AR42J cells by peptide inhibitors.

[0022]FIG. 6 is a bar graph of the cellular distribution ofimmunogold-labeled AR4-2J tumor cells.

[0023]FIG. 7 is a photograph of a Western blot analysis of proteinextracts from nuclear membranes of adenocarcinoma cells using antibodiesraised against Peptide 1.

[0024]FIG. 8 is a photograph of a Western blot analysis of proteinextracts from extranuclear and plasma membranes of adenocarcinoma cellsusing antibodies raised against Peptide 1.

[0025]FIG. 9 is a plot graph illustrating the C170HM2 tumor weight ofcontrol and anti-CCK-B/gastrin receptor-treated animals.

[0026]FIG. 10 is a plot graph illustrating the cross-sectional area ofC170HM2 tumors from control and anti-CCK-B/gastrin receptor-treatedanimals.

[0027]FIG. 11 is a bar graph showing the mean C170HM2 tumor weights ofcontrol and anti-CCK-B/gastrin receptor-treated animals.

[0028]FIG. 12 is a bar graph showing the mean cross-sectional area ofC170HM2 tumors of control and anti-CCK-B/gastrin receptor-treatedanimals.

[0029]FIG. 13 is a bar graph showing the mean number of C170HM2 tumorsin control and anti-CCK-B/gastrin receptor-treated animals.

[0030]FIG. 14 is a bar graph showing the median C170HM2 tumor weight ofliver metastases, of control and anti-CCK-B/gastrin receptor-treatedanimals.

[0031]FIG. 15 is a bar graph showing the median cross-sectional area ofC170HM2 tumors from control and anti-CCK-B/gastrin receptor-treatedanimals.

[0032]FIG. 16 is a bar graph showing the median C170HM2 tumor number incontrol and anti-CCK-B/gastrin receptor-treated animals.

[0033]FIG. 17 is a bar graph showing the mean and median liver C170HM2tumor number in control and anti-CCK-B/gastrin-receptor-treated animals.

[0034]FIG. 18 is a bar graph showing the mean and median liver C170HM2tumor weight in control and anti-CCK-B/gastrin-receptor-treated animals.

[0035]FIG. 19 is a bar graph showing the mean and median values for thecross-sectional area of C170HM2 liver tumor metastases in control andanti-CCK-B/gastrin-receptor-treated animals.

[0036]FIG. 20 depicts a graph showing the concentration of radiolabeled¹²⁵I-antibodies in C170HM2 liver tumor xenografts of control (normalrabbit serum) and anti-GRP1-treated nude mice.

[0037]FIG. 21 depicts a bar graph showing the mean C170HM2 liver tumornumber per liver of xenografts of control and anti-GRP1-treated nudemice.

[0038]FIG. 22 depicts a bar graph showing the mean C170HM2 liver tumorweight of liver xenografts of control and anti-GRP1-treated nude mice.

[0039]FIG. 23 depicts Western blots of C170HM2 liver tumor xenograftproteins of control and anti-GRP1-treated nude mice.

[0040]FIG. 24 is a photograph of a histological section taken with alight microscope showing a hematoxylin/eosin-stained section of aC170HM2 liver xenograft of a control mouse.

[0041]FIG. 25 is a photograph of a histological section taken with alight microscope showing a hematoxylin/eosin stained section of aC170HM2 liver xenograft from a mouse treated with rabbitanti-GRP1antibodies.

DETAILED DESCRIPTION OF THE INVENTION

[0042] The methods of the invention are directed to the treatment ofgastrin hormone-dependent tumors in animals, including humans, andcomprise administering to a patient an anti-CCK-B/gastrin-receptorimmunogen, which produces antibodies in the immunized patient which bindto the CCK-B/gastrin-receptor on the tumor cells, so as to prevent thebinding of the hormone to the receptor in order to inhibit thegrowthpromoting effects of the hormone. More importantly, from aclinical point of view, the receptor/anti GRP1 complex is rapidlyinternalized, traverses the cytoplasm and enters the nucleus. Thisapparently triggers the affected tumor cells to commit suicide(apoptosis).

[0043] The immunogens comprise natural or synthetic peptides of thehuman CCK-B/gastrin-receptor which act as immunomimics. In particular,two synthetic peptides have been developed as the immunomimics. Thesepeptides, developed from the amino acid sequence of theCCK-B/gastrin-receptor, are immunogenic and cross-reactive with theendogenous CCK-B/gastrin-receptor of tumor cells both in vivo and invitro. Peptide 1 consists of amino acids 5 through 21 of the CCK-B/gastrin-receptor sequence: KLNRSVQGTGPGPGASL (Peptide 1, SEQ ID NO.: 1in the Sequence Listing). Peptide 1 constitutes the amino-terminal endof the receptor and is located on the extracellular surface of the cellmembrane (see FIG. 1).

[0044] In another embodiment, the immunogen comprises Peptide 4, whichconsists of the following amino acid sequence of theCCK-B/gastrin-receptor: GPGAHRALSGAPISF (Peptide 4, SEQ ID NO.: 2 in theSequence Listing). Peptide 4 is part of the fourth extracellular domainof the receptor and it too is on the outer side of the plasma membrane(see FIG. 1).

[0045] The immunogens may also comprise an extension or spacer peptidesuitable for projecting the immunomimic peptide away from the proteincarrier and enhancing its capacity to bind the lymphocyte receptors. Asuitable spacer peptide sequences the amino acid sequence SSPPPPC(Serine (Ser) spacer, SEQ ID NO.:3 in the Sequence Listing). Howeverother spacer peptides would be suitable as well. The immunomimicpeptides, with or without the spacer, are then conjugated to a proteincarrier, such as Diphtheria toxoid, via a cysteine residue at thecarboxy terminal end. The spacer peptides are not immunologicallyrelated to the CCK-B/gastrin-receptor-derived peptides and shouldtherefore enhance, but not determine, the specific immunogenicity of thereceptor-derived peptides.

[0046] The presence and density of CCK-B/gastrin-receptors on tumorcells in a patient can be determined by reacting labeled anti-receptorantibodies with a sample of obtained from tumor biopsy sample. Theanti-receptor antibodies can be labeled with either a radioactivetracer, a dye or a fluorescent label. In addition, the responsiveness ofthe tumor cells to gastrin can be evaluated in vitro from a tumor biopsysample of the patient using standard techniques. Patients having tumorswith biopsy samples positive for the CCK-B/gastrin-receptor antibodyassay are typical candidates for treatment by the methods of theinvention.

[0047] An effective dosage ranging from 0.001 to 2 mg of the immunogeniccomposition is administered to the patient for the treatment of thegastrointestinal cancer. The effective dosage of the immunogeniccomposition should be capable of eliciting an immune response in apatient consisting of effective levels of antibody titer against theCCK-B/gastrin-receptor 1-3 months after immunization. Following theimmunization of a patient, the effectiveness of the immunogens ismonitored by standard clinical procedures, such as ultrasound andmagnetic resonance imaging (MRI), to detect the presence and size oftumors. The antibody titer levels against the receptor may also bemonitored from a sample of blood taken from the patient. Boosterimmunizations should be given as required to maintain an effectiveantibody titer. Effective treatment of gastrin-dependent cancers, suchas stomach, liver, pancreatic and colorectal adenocarcinomas, accordingto this method should result in inhibition of tumor growth and adecrease in size of the tumor.

[0048] The antibodies raised by the anti-CCK-B/gastrin-receptorimmunogens of the present invention may have anti-trophic effectsagainst gastrin-dependent tumors by three potential mechanisms: (i)inhibition of gastrin binding to its receptor, (ii) degradation ordisruption of the signal transduction pathway of tumor cellproliferation; and (iii) induction of apoptosis (or cell suicide) incells where receptor/antibody complexes are internalized and migrateinto the nucleus.

[0049] In another embodiment of the invention,anti-CCK-B/gastrin-receptor antibodies are administered to a patientpossessing a CCK-B/gastrin-receptor-responsive tumor. The antibodiesspecifically bind to the CCK-B/gastrin-receptors on the tumor cells. Thebinding of the antibodies to the receptors prevents the binding ofgastrin to its ligand in the membranes of cells and, therefore, thegrowth signal for the gastrin-dependent tumor cells is inhibited and thegrowth of the tumor is arrested. The antibodies are preferably chimericor humanized antibodies, or fragments thereof, which effectively bind tothe target receptor and may be produced by standard techniques such asthose disclosed in U.S. Pat. Nos. 5,023,077, 5,468,494, 5,607,676,5,609,870, 5,688,506 and 5,662,702, the disclosures of which are herebyincorporated by reference. These exogenously produced antibodies mayalso be useful for killing tumor cells that bear theCCK-B/gastrin-receptor on their plasma membranes by virtue of theirinhibiting the growth of the tumor cells or delivering a toxic substanceto the tumor cell. Preferred anti-CCK-B/gastrin antibodies for therapyare those reactive with extracellular domains 1 and 4 of the receptorprotein shown in FIG. 1 as GRP-1 and GRP-4, respectively. Particularlypreferred are, antibodies which specifically recognize and bind aminoacid sequences of the receptor protein corresponding to Peptides 1 and4. The inhibition of tumor growth in this method of immunization is alsomonitored by ultrasound imaging and MRI and repeated immunizations areadministered as required by the patient.

[0050] The effectiveness of the antibodies in inhibiting tumor cellgrowth and killing of tumor cells can be enhanced by conjugatingcytotoxic molecules to the anti-CCK-B/gastrin antibodies. The cytotoxicmolecules can be toxins, for example, cholera toxin, ricin, α-amanitin,or radioactive molecules labeled, for example with ¹²⁵I or ¹³¹I, orchemotherapeutic agents, for example, cytosine arabinoside or5-fluorouridine.

[0051] In addition to antibodies radiolabeled with ¹²⁵I and ¹³¹I, theanti-CCK-B/Gastrin-receptor antibodies can also be labeled withradionuclide such as ¹¹¹Indium and ⁹⁰Yttrium. In this aspect of theinvention the antibodies are useful for the detection and diagnosing ofCCK-B/gastrin-receptor possessing tumors in vivo, by administering theseantibodies to the patient, and detecting bound antibodies onCCK-B/gastrin-receptor-containing tumor cells. After allowing theradio-labeled anti-CCK-B/gastrin antibodies to reach the tumor, about1-2 hours after injection, the radioactive, “hot spots” are imaged usingstandard scintigraphic procedures as previously disclosed (Harrison'sPrinciples of Internal Medicine, Isselbacher et al. eds. 13^(th) Ed.1994).

[0052] The compositions in which the immunogens are administered for thetreatment of gastrin-dependent tumors in patients may be in a variety offorms. These include, for example, solid, semi-solid and liquid dosageforms, such as powders, liquid solutions, suspensions, suppositories,and injectable and infusible solutions. The preferred form depends onthe intended mode of administration and therapeutic applications. Thecompositions comprise the present immunogens and suitablepharmaceutically acceptable components, and may include other medicinalagents, carriers, adjuvants, excipients, etc. Suitable adjuvants mayinclude nor- muramyl dipeptide (nor-MDP, Peninsula Labs., Calif.), andoils such as Montanide ISA 703 (Seppic, Inc., Paris, France), which canbe mixed using standard procedures. Preferably, the compositions are inthe form of unit dose. The amount of active compound administered forimmunization or as a medicament at one time, or over a period of time,will depend on the subject being treated, the manner and form ofadministration, and the judgment of the treating physician.

[0053] The anti-CCK-B/gastrin-receptor antibodies of the invention forpassive immunization are preferably administered to a patientintravenously using a pharmaceutically acceptable carrier, such as asaline solution, for example, phosphate-buffered saline.

EXAMPLE 1

[0054] Preparation of GRP1-DT and GRP4-DT Conjugates

[0055] CCK-B/gastrin-receptor peptides were prepared by standard solidstate peptide synthesis. To make immunogens capable of inducing specificimmune responses each of Peptide 1 and 4 was synthesized containing thespacer sequence SSPPPPC (SEQ ID NO.:3 in the Sequence Listing) at itscarboxy terminus. These peptides were subsequently conjugated to aminogroups present on the carrier, Diphtheria toxoid (“DT”), via theterminal peptide amino acid residue cysteine of the spacer utilizing aheterobifunctional linking agent containing a succinimidyl ester at oneend and maleimide at the other end of the linking agent by either ofMethod A or Method B as described below.

[0056] Method A: As previously described in U.S. Pat. No. 5,023,077, thelinking of Peptide 1 or 4 above and the carrier is accomplished asfollows. Dry peptide was dissolved in 0.1 M Sodium Phosphate Buffer, pH8.0, with a thirty-fold molar excess of dithiothreitol (“DTT”). Thesolution was stirred under a water saturated nitrogen gas atmosphere forfour hours. The peptide containing reduced cysteine was separated fromthe other components by chromatography over a G10 Sephadex columnequilibrated with 0.2 M acetic acid. The peptide was lyophilized andstored under vacuum until used. The carrier was activated by treatmentwith the heterobifunctional linking agent e.g. Epsilon- maleimidocaproicacid N-hydroxysuccinimide ester, (“EMCS”), in proportions sufficient toachieve activation of approximately 25 free amino groups per 105molecular weight of carrier. In the specific instance of diphtheriatoxoid, this amounted to the addition of 6.18 mg of EMCS (purity 75%) toeach 20 mg of diphtheria toxoid.

[0057] Activation of diphtheria toxoid was accomplished by dissolvingeach 20 mg aliquot of diphtheria toxoid in 1 ml of 0.2 M SodiumPhosphate Buffer, pH 6.45. Aliquots of 6.18 mg EMCS were dissolved into0.2 ml of Dimethyl Formamide (“DMF”). Under darkened conditions, theEMCS was added dropwise in 50 microliter (“μl”) amounts to the DT withstirring. After 2 hours of incubation in darkness, the mixture waschromatographed on a G50 Sephadex column equilibrated with 0.1 M SodiumCitrate buffet, pH 6.0, containing 0.1 mM EDTA.

[0058] Fractions containing the EMCS activated diphtheria toxoid wereconcentrated over a PM 10 ultrafiltration membrane under conditions ofdarkness. The protein content of the concentrate was determined byeither the Lowry or Bradford methods. The EMCS content of the carrierwas determined by incubation of the activated carrier with cysteine-HClfollowed by reaction with 10 mM of Ellman's Reagent 5,5′dithio-bis(2-nitrobenzoic acid) 10 mM. The optical density difference between ablank tube containing cysteine-HCl and the sample tube containingcysteine-HCl and carrier was translated into EMCS group content by usingthe molar extinction coefficient of 13.6×10³ for 5-thio-2-nitrobenzoicacid at 412 nm.

[0059] The reduced cysteine content (—SH) of the peptide was alsodetermined utilizing Ellman's Reagent. Approximately 1 mg of peptide wasdissolved in 1 ml of nitrogen gas saturated water and a 0.1 ml aliquotof this solution was reacted with Ellman's Reagent. Utilizing the molarextinction coefficient of 5-thio-2-nitrobenzoic acid (13.6×10³, the freecysteine —SH was calculated. An amount of peptide containing sufficientfree —SH to react with each of 25 EMCS activated amino groups on thecarrier was dissolved in 0.1 M Sodium Citrate Buffer, pH 6.0, containing0.1 mM EDTA, and added dropwise to the EMCS activated carrier underdarkened conditions. After all the peptide solution had been added tothe carrier, the mixture was incubated overnight in the dark under awater-saturated nitrogen gas atmosphere.

[0060] The conjugate of the peptide linked to the carrier via EMCS wasseparated from other components of the mixture by chromatography over aG50 Sephadex column equilibrated with 0.2 M Ammonium Bicarbonate. Theconjugate eluted in the column void volume was lyophilized and storeddesiccated at 20° C. until used.

[0061] The resulting conjugate may be characterized as to peptidecontent by a number of methods known to those skilled in the artincluding weight gain, amino acid analysis, etc. Conjugates of Peptides1 and 4 with spacer and diphtheria toxoid produced by this method weredetermined to have an effective peptide/carrier ratio of 5-35 moles ofpeptide per 100 KD MW of carrier and all were considered suitable asimmunogens for immunization of test animals. Preferably, the range ofthe peptide from 10-30 moles per 100 KD MW of DT produced an effectiveimmune response.

[0062] Method B: In a preferred method, conjugates comprising GRP1coupled to DT and GRP4 peptide coupled to DT were prepared at roomtemperature as follows. Purified DT (400 mg) was dissolved in 20 ml of0.5 M phosphate buffer, pH=6.6, saturated with nitrogen gas to give a DTsolution of 20 mg/ml. The DT solution was placed in a 60 ml dark amberglass bottle (serving as a reaction vessel and filtration reservoir).EMCS coupling reagent (123.6 mg) was dissolved in 2.0 ml ofdimethylformamide. The EMCS solution was added dropwise to the DTsolution over a 15 minute period with continuous stirring. The bottlewas capped, and the mixture was stirred at room temperature for anadditional 1 hour 45 minutes, to form activated DT (M-DT). The M-DT wasthen purified by diafiltration using an Amicon Model TFC10 Thin-ChannelUltrafiltration System per operating manual 1-113G with a XM50 diaflowultrafiltration membrane. The M-DT was washed twice against volumes of420 ml phosphate buffer, concentrating to 20 ml each time, then washedonce against 420 ml of 0.1 M sodium citrate buffer, pH=6.0, containing0.1 M EDTA, and concentrating the solution down to 20 ml.

[0063] To make GRP1-DT conjugate, 2.02 ml of M-DT solution (containing22.3 mg M-DT) was placed in a 10 ml dark amber glass vial, then 13 mg ofGRP1 peptide was dissolved in the citrate buffer to give 40 mg/mlpeptide and added dropwise to the M-DT solution with stirring. To makeGRP4-DT conjugate, 2.21 ml of M-DT solution (containing 24.4 mg M-DT)was placed in a 10 ml dark amber glass vial, then 13 mg of GRP4 peptidewas dissolved in the citrate buffer to give 40 mg/ml peptide and addeddropwise to the M-DT solution with stirring.

[0064] The reactions were allowed to proceed overnight in the dark. Eachconjugate was removed from the reaction vessels and separately dialyzedin 12,000-14,000 MW cutoff dialysis tubing against 5 changes 500 ml of0.1 M ammonium bicarbonate solution. Each conjugate was lyophilized. Theconjugates were then analyzed by amino acid analysis and their peptideto DT substitution ratios were determined to be 21.8 peptides per 10⁵ MWof DT for GRP1-DT and 21.1 peptides per 10⁵ MW of DT for GRP4-DT.

[0065] Conjugates of Peptides 1 and 4 with spacer and DT produced bythis method have an effective peptide/carrier ratio of 5-35 moles ofpeptide per 100 KD MW of carrier and all are considered suitable asimmunogens. A preferred ratio range for producing an effective immunerespose is from 10-25 moles of peptide per 100 KD MW of DT.

[0066] Preparation of Immunogens

[0067] The present immunogens containing either Peptide 1 or Peptide 4with spacer conjugated to DT as described above were used to immunizerabbits. Immunogens were prepared as follows: Conjugate was dissolved in0.15 M Sodium phosphate buffered saline, pH 7.3 to a concentration of3.79 mg/ml. The conjugate solution was added to Montanide ISA (703)Adjuvant (Seppic, Inc.) in a 30:70 (wt:wt) ratio of conjugate solutionto Montanide ISA 703, then the mixture was homogenized using a SilversonHomogenizer for 3 minutes at 8,000 RPM to form an emulsion containing 1mg/ml of conjugate.

[0068] Immunization and Sample Collection

[0069] Rabbits were injected intramuscularly with 0.1 ml of immunogenconsisting of 0.1 mg of either GRP1-DT, or GRP4-DT conjugate. Eachrabbit was given injections of immunogen at 0 and 4 weeks. Blood wascollected from each rabbit at 6 and 8 weeks of the experiment. Serum wasprepared from each blood sample and stored at −20° C. until utilized inassays to determine the presence of anti-CCK-B/gastrin-receptorantibodies.

[0070] Enzyme-Linked Immunosorbent Assay (ELISA)

[0071] A solid-phase ELISA was used to screen for reaction orcross-reaction of antisera raised against Peptide 1 and Peptide 4 ofeach immunized rabbit. The ELISA was carried out by coating polystyrene96 well plates (IMMULON II, Dynatech) with 25 μl/well of 10 μg/ml ofPeptide 1 linked to bovine serum albumin (BSA) (“GRP1-BSA”), or Peptide4 linked to BSA (“GRP4-BSA”) antigen in 0.1 M Glycine-HCl, pH 9.5buffer. The plates were incubated overnight at 4° C., and subsequentlywashed in buffer.

[0072] Antisera obtained from the immunized rabbits were seriallydiluted to a range of 10⁻¹ to 10⁻⁸ in 1% BSA-FTA hemagglutinationbuffer, pH 7.2. Twenty five μl of test antiserum per well was incubatedwith each test peptide for 1 hr at room temperature. After incubation,the plates were washed thoroughly with buffer to remove any unboundantibody. Each well was treated with 25 μl of biotinylated goatanti-rabbit IgG (H+L) diluted 1: 1000 in 1% BSA-FTA dilution buffer for1 hour at room temperature. After washing the plates to remove unboundanti-rabbit reagent, each well was incubated for 1 hour at roomtemperature with 25 μl of avidin-alkaline phosphatase conjugate diluted1:1000 in 1% BSA-FTA buffer. The plates were washed thoroughly to removeunbound avidin-alkaline phosphatase reagent, and incubated with 25 μl of1 mg/ml of p-nitrophenylphosphate (“PNPP”) in 10% diethanolamine buffercontaining 0.01% MgCl_(2.6)H₂O, pH 9.8. The plates were allowed todevelop until the absorbance of the reaction at 490 nm wavelengthreached an optical density between 0.8 to 1.5. To test the specificityof the antisera produced by the rabbits, rabbits were also immunizedwith DT and for ELISA testing, plates were coated with DT as antigen todetermine the reactivity of the antisera produced against the carrier.

[0073]FIG. 2 shows the ELISA results using Peptide 1/GRP1 and FIG. 3shows the ELISA results using Peptide 4/GRP4 as the antigen. As seen inFIG. 2, the ELISA results show that the rabbits immunized with Peptide1-spacer-DT conjugate produced high antibody titers which specificallybind to Peptide 1, as indicated by the antibody binding Peptide 1 evenat high (1: 100,000) dilutions of the antiserum. Similarly, FIG. 3 showsthat rabbits immunized with Peptide 4-spacer-DT conjugate produced hightiters of anti-Peptide 4 antibodies. As seen in FIGS. 2 and 3, therabbits immunized against each peptide produced antibodies which boundspecifically to each peptide at small antisera concentration. The dataindicate that the anti-Peptide 1 and anti-Peptide 4 antibodies have alarge capacity for binding Peptides 1 and 4 of theCCK-B/gastrin-receptor. The data also shows that immunization of rabbitswith the present conjugates elicits powerful immune responses againstPeptide 1 and Peptide 4, respectively. In addition, rabbits immunizedwith either Peptide-1 or Peptide-4 conjugate appeared and behaved normaland did not exhibit any symptoms of disease or pathologies during theexperiments.

EXAMPLE 2

[0074] The following experiments were performed to establish thespecificity of antibodies raised in rabbits against the GRP1-DT peptidecontaining Ser spacer described Example 1 using Method B. A series oftests were conducted to assess the specificity of rabbit antibodiesinduced by immunization with the GRP1-DT and affinity purified byimmunoadsorption over a GRP1-Ser Sepharose column.

[0075] An inhibition ELISA was used to assess the specificity of theaffinity purified antibodies for GRP1-Ser peptide. The assays were runas follows: GRP1-Ser-BSA conjugate was coated onto 96 well plates(Immulon U bottom) by overnight incubation of 50 μl of a 2 μg/mlsolution of conjugate in glycine buffer (0.1M, pH=9.5) at 4° C. Affinitypurified anti-GRP1 Ab (at a final concentration of 10 ng/ml) wascombined with various inhibitors (in 1:10 dilution series) and incubatedfor 1 hour at room temperature. The inhibitors included GRP1-Ser, GRP1EPT, Ser, human gastrin 17(1-9)-Ser spacer (hG17(9)-Ser), GRP1 EPT+Ser,and buffer (no inhibitor). Incubation buffer consisted of PBS+0.5%BSA+0.05% Tween 20+0.02% NaN₃. Subsequent steps used the same bufferwithout BSA. The 96 well plates were washed free of nonboundGRP1-Ser-BSA, and the Ab+inhibitor mixtures were added (50 μl/well).After 1 hour, the plates were washed and a goat anti-rabbit Ig (H+L)alkaline phosphatase conjugate (Zymed) was added (1:2000 dilution).After 1 hour incubation, the plates were washed to remove nonboundreagent, and 50 μl/well of pNPP substrate (Sigma) solution (1 mg/ml) wasadded in substrate buffer (PBS+0.1 mg/ml MgCl₂₊₁₀% diethanolamine+0.02%NaN₃). Following a 60 minute incubation, absorbance was measured on aMRX reader (Dynatech Laboratories). Samples were run in duplicate, andmeans were calculated for each concentration. Background binding(established with affinity purified rabbit anti-GnRH antibodies) wassubtracted from all values, and the % Inhibition relative to noinhibitor added (anti-GRP1 Ab+buffer) was calculated for each inhibitortested: %Inhibition=(100)(1-((A_(uninhibited)−A_(inhibited))/A_(uninhibited)),where A=Absorbance. The results are shown in FIG. 4.

[0076]FIG. 4 presents the percent inhibition of antibody binding as afunction of inhibitor concentration. As can be seen in the figure, theGRP1-Ser peptide fully inhibited antibody binding to GRP1-Ser-BSA.Approximately 60% inhibition was attained with the GRP1 EPT peptide,which does not contain the Ser spacer sequence, and by an equimolarmixture of GRP1 EPT plus Ser spacer. The failure of these peptides toproduce full inhibition suggests that a proportion of the antibodieswere specific for an epitope(s) comprising elements of both the GRP1 andthe Ser spacer sequences. No inhibition was obtained by either the Serspacer sequence itself or by an unrelated peptide bearing the Ser spacer(“hG17(9)-Ser”, consisting of the amino-terminal nine residues ofhG17followed by the Ser spacer). These ELISA results demonstrate thatthe affinity purified antibody preparation was specific for the GRP1-Serpeptide, and that 60% of the binding activity was directed against thegastrin-receptor epitope component of the peptide.

EXAMPLE 3

[0077] AR42J tumor cells (European Collection of Animal Cell cultures,Porton Down, UK) are derived from a rat pancreatic adenocarcinoma andare known to have well characterized CCK-B/gastrin-receptors. Thus AR42Jwere tested to confirm the expression of CCK-B/gastrin-receptor andspecificity of the receptor for hG17 by radioligand inhibition. AR42Jcells were cultured at 37° C. with 7% CO₂ in complete RPMI 1640 (Sigma)supplemented with 10% FCS (Gemini Bioproducts), 2 mM glutamine (JRHBiosciences), 1 mM sodium pyruvate (JRH B.) and 50 μg/ml gentamicin(Gemini Bioproducts). The cells were harvested from 175 cm² T-flasks(Falcon Plastics) with PBS containing 0.25% EDTA, then washed twice withPBS (no EDTA) by centrifugation (400 X g for 10 min). The cells werekept at 0-4° C. for all manipulations. A single cell suspension wasprepared in buffer, and the cell concentration was adjusted to 10⁶cells/ml. Aliquots of 1 ml of cell suspension were added to 12×75 mmculture tubes, then the cells were centrifuged and the supernatantsdiscarded. The cells were resuspended in PBS (0.1 ml/tube) containinghuman G17 (hG17), gonadotropin releasing hormone (GnRH), or no peptide.The peptide concentrations were 1.0 ng/ml, 100 ng/ml and 10 μg/ml. Analiquot of 0. 1 ml of ¹²⁵I-hG17 (NEN), containing approximately 26,300CPM (specific activity, 2200 Ci/mmol), was added to each tube. The tubeswere vortexed, then incubated for 15 minutes. The cells were washedtwice with PBS, then counted in a y counter (Wallac). Samples were runin duplicate. Background counts were subtracted, then the % inhibitionof ¹²⁵I-hG17 binding by each inhibitor was calculated using theequation: %Inhibition=(100)(1-((CPM_(uninhibited)−CPM_(inhibited))/CPM_(inhibited)).

[0078] The results of the radioligand binding inhibition tests are shownin FIG. 5, which presents the means (±SE) of the individual values. Ascan be seen in the figure, binding of ¹²⁵I-hG17 to AR42J cells wasinhibited by hG17. The degree of inhibition increased with the quantityof inhibitor added, to 32% inhibition at 1 μg hG17 per tube, the highestconcentration of peptide tested. Conversely, GnRH produced no inhibitionat the two highest concentrations tested (the 6% inhibition obtainedwith 100 pg GnRH was considered to be nonspecific), indicating that theinhibition by hG17 was specific for gastrin. These results confirmed thecell surface expression of gastrin-receptor by the AR42J tumor cells.

EXAMPLE 4

[0079] Binding of the GRP1-Ser specific antibodies to AR42J cells wasassessed by immunofluorescence. AR42J cells were grown as in theprevious Examples and harvested with cell scrapers from 175 cm² T-flasksand washed twice with buffer (PBS with 0.02% NaN₃) by centrifugation(400 X g for 7 min). The cells were kept at 0-4° C. for allmanipulations. A single cell suspension was prepared in buffer, and thecell concentration was adjusted to 10⁶ cells/ml. The cell suspension wasadded to 1.5 ml microfuge tubes (1 ml/tube). The cells were pelleted bycentrifugation and supernatants were aspirated. The cells wereresuspended in buffer (0.1 ml/tube) containing peptide inhibitors (1.0mg/ml). The inhibitors included GRP1-Ser, GnRH, hG17(9)-Ser and buffer(no inhibitor). Antibodies, including the rabbit anti-GRP1-Ser (100μg/ml), affinity purified rabbit anti-DT (negative control, 100 μg/ml),mouse anti-AR42J antiserum (positive control, 1:100 dilution, heatinactivated) or normal mouse serum were added to the appropriate tubesand the contents were mixed. The cells were incubated for 1 hour, withoccasional mixing. The cells were then washed three times with buffer,and 0.1 ml of fluorescein-labeled goat anti-rabbit IgG (AntibodiesIncorporated) (diluted 1:50) was added per tube. The cells treated withmouse sera were developed with a fluorescein-anti-mouse IgG reagent(Zymed). The cells were re-suspended by vortexing, then incubated for 1hour. The cells were again washed three times, then re-suspended inglycerol: PBS (1:1, v:v), 50 μl/tube. Wet mounts were prepared with thecontents of each tube, and the cells examined using a Laborlux 12fluorescent microscope (Leitz). Fluorescence was scored on a scale of 0to 4, with 0 representing background fluorescence (obtained with thenormal mouse serum) and 4 representing maximal fluorescence (obtainedwith the mouse anti-AR42J positive control antiserum).

[0080] The results of the immunofluorescesce tests are presented inTable 1. As can be seen in the Table, AR42J cells treated withanti-GRP1-Ser antibodies in the absence of peptide inhibitors fluorescedstrongly, indicating that the antibody bound to the cells. Rabbitanti-DT antibodies did not produce fluorescent staining, demonstratingthat the staining observed with the anti-GRP1-Ser antibodies was not aconsequence of non-specific cell surface binding by rabbitimmunoglobulin. Moreover, the binding was shown to be specific for theGRP1-Ser peptide. Addition of GRP1-Ser fully inhibited binding, whereasunrelated peptides, including hG17(9)-Ser and GnRH, failed to inhibit.As the GRP1 epitope comprises residues 5-21 of the gastrin-receptor, itwas concluded that the anti-GRP1-Ser antibodies were specific for thegastrin-receptor expressed by AR42J cells. TABLE 1 Antibody InhibitorPreparation GRP1-Ser hG17(9)-Ser GnRH Buffer Rabbit anti-GRP1-Ser   0  3+   2+   3+ Rabbit anti-DT 0.5+ 0.5+ 0.5+ 0.5+ Mouse anti-AR42J   4+Normal Mouse Serum   0

EXAMPLE 5

[0081] AR42J cells, passage nos. 16-18 were cultured in RPMI-1640 mediumcontaining 10% FCS and 2 mM glutamine. All cells were maintained at 37°C. in 5% CO₂ in air at 100% humidity, grown to 80% conflucency in T75flasks (Falcon, London, UK) and passage following a 0.02% EDTA treatmentto bring adherent cells into suspension. Cells were incubated for 10, 30seconds, 30 minutes and 1 hour with anti-CCK-B/gastrin-receptor antibody(aGR) generated in rabbits with a CCK-B/gastrin Peptide 1 receptorimmunogen of the invention as described in Example 1, which had beenpurified by affinity chromatography in a column prepared with Peptide 1.

[0082] The cells were fixed in 1% glutaraldehyde for one hour andprepared for immunoelectron microscopy (ImmunoEM) studies using standardtechniques. The cell suspensions was centrifuged twice at 2000 rpm for 2minutes and then the cell pellet resuspended in phosphate bufferedsaline (PBS). The cell pellet was infiltrated with LRwhite plasticresin. Ultrathin sections of 70-90 nm in thickness were cut and place onPioloform coated nickel grids. The grids were placed in normal goatserum (Dako, High Wycombe, UK) in 0.1% bovine serum albumin (BSA)(Sigma, Poole, Dorset) and incubated at room temperature for 30 minutes.Grids were rinsed in PBS then incubated with a secondary antibody,biotin-conjugated goat anti-rabbit antibody (gold-labelled), diluted1:50 in 1% BSA, for 1 hour at room temperature. Control experiments wereperformed without secondary antibody. After final PBS wash, the gridswere counterstained in saturated aqueous uranyl acetate for 3 minutesand Reynold's lead citrate for 3 minutes. Gold particles on the cellmembrane, in the cytoplasm, on the nuclear membrane and within thenucleus were counted. Twenty-five cells/grid were counted by anindependent observer. For controls AR42J cells were exposed toantibodies for less than 1 second, and liver cells which are devoid ofCCK-B/gastrin-receptor were used. AR42J cells exposed to normal IgG werealso used as controls for determining non-specific binding of theanti-CCK-B/gastrin-receptor antibodies. The results of these experimentsare shown in Table 2 and FIG. 6. TABLE 2 Distribution ofCCK-B/gastrin-receptor Immunogold Particles Within AR42J cells Cell CellNuclear Nuclear membrane matrix membrane matrix No. gold 14.2(±0.97)43.3(±2.32) 9.3(±0.81) 51.4(±3.32) particles Percent 12% 36.6% 7.9%43.5% distribution within cell

[0083] As demonstrated in Table 2 and FIG. 6, immunogold-antibodyparticles attached to the CCK-B/gastrin-receptor were localized onplasma membrane, cytoplasm, nuclear membrane, and nuclear matrix of theadenocarcinoma cells, further demonstrating that the antibody/receptorcomplex is internalized by the cells.

[0084] As seen in Table 2, the immunoEM studies using an antiserumdirected against the amino-terminal end of the CCK-B/gastrin-receptorshows that after one hour incubation, the distribution ofimmunogold-labelled CCK-B/gastrin-receptor antibody is quicklyinternalized as 12% of the antibody receptor complex is associated withthe cell membrane, 36.6% is within the cytoplasm, 7.9% is in the nuclearmembrane and, quite surprisingly, 43.5% is within the cell nucleus.Areas of intense CCK- B/gastrin-receptor immunoreactivity within thenucleus are found on chromatin, which may suggest specific binding sitesfor regulation of the DNA.

[0085] These electron microscopy studies with anti-immunoglobulinconjugated to gold beads (immmunogold) reveal that an extremely rapidturnover of the anti-receptor/receptor complex occurs in the tumorcells; as early as 10 seconds after exposure to antibodies, complexesare detectable in the cell nucleus as seen in FIG. 6.

EXAMPLE 6

[0086] Adenocarcinoma cell lines, namely AR42J, HCT116, C170HM2, LoVo,ST16 and MGLVA1, were grown in vitro and harvested as described inExamples 3. Cells from 30×T-75 flasks were suspended in 5 ml ofhomogenization buffer (1 mM sodium hydrogen carbonate, 2 mM magnesiumchloride, 1 nM phenylmethylsulphonyl fluoride, 40 mM sodium chloride, 10μl leupeptin, 1 μM pepstatin, 5 nM EDTA [Sigma]). Homogenization wascarried out by 5 bursts of 5 second duration in a homogenizer. Forextranuclear membranes, tissue debris was pelleted by centrifugation at500g, 7 minutes, 4° C. The pellet was discarded and the supernatantcentrifuged at 500g, 4° C. to remove further debris. The supernatant wasrecentrifuged at 48,000g, 1 hour, 4° C. The pellet containing theextranuclear membrane preparation was suspended in Tris/NP-40 solution(0.1 M TRIZMA, 0.5% NONIDET P40 [Sigma Chemical]).

[0087] For nuclear membrane preparations, following homogenization in asecond homogenization buffer (25 mM Tris-HCl, pH 7.4, 0.1% TRITON 100,0.32 M sucrose, 3 mM MgCl₂, 2 mM EGTA, 0.1 mM sperminetetrahydrochloride, 2 mM PMSF, 10 mM bezomidine hydrochloride, 3 mM EGTAaminoacetonitrile hydrochloride [Sigma]), tissue debris was pelleted bycentrifugation at 400 g, for 10 minutes at 4° C. The pellet wasresuspended in 55% (0.2 M) sucrose in HPLC water. This mixture was spunat 60,000 g for 1 hour at 4° C. The pellet was washed with 0.4% NONIDETP40 in homogenization buffer without TRITON 100. The pellet was spun at700 g for 15 min at 4° C. and resuspended in homogenization bufferwithout TRITON 100.

[0088] Protein content is determined by the Lowry method (using a kitfrom Pierce). Samples containing 10-15 μg of protein were loaded onto a8-16% Tris/glycine gradient polyacrylamide gel electrophoresis PAGE(Novex R and D systems) in Tris/glycine buffer and run for 90 minutes at125 constant volts, 36 mA. The gel was fixed in 10% glacial acetic acidfor 1 hour and samples were blotted onto nitrocellulose membrane. Themembranes were incubated in 1% BSA for 1 hour, followed by incubationwith GRP-1 antiserum (with and without preabsorption) for 1 hour.Antibody binding were detected by the avidin:biotin-peroxidase complexmethod using diamino-bezidene as the substrate. The Western blotanalysis results using Rabbit-antiserum raised against Peptide 1 (Rabbitanti-GRP1 antiserum) are shown in FIG. 7 and FIG. 8.

[0089] As shown in FIG. 7, the protein molecular weight markers rangefrom 116, 66, 45 and 29 kDa. The blot shows a prominent anti-Peptide 1immunoreactive band localizing at about 43 kDa in all adenocarcinomacells studied, i.e., HCT116, C170HM2, LoVo, ST16 and MGLVA1, except one(AP5LV). This protein corresponds to a truncated form of theCCK-B/gastrin-receptor. Some cell lines (HCT 116 and C170HM2) show atleast 3 other bands, ranging in molecular weight between 60 and 100 KDa.The data indicate that the anti-CCK-B/gastrin-receptor antibodies canrecognize and bind to various isoforms of the CCK-B/gastrin-receptor intumor cells.

[0090]FIG. 8 shows a Western blot from extranuclear (ENM) and plasmamembrane of C170HM2 and HCT 116 adenocarcinoma cells. As shown in FIG.8, adenocarcinoma cell lines tested for ENM CCK-B/gastrin-receptorsdemonstrate the existence of two strongly stained bands: one about 43KDa and the other at about 66 KDa. When only the plasma membranefraction was stained, a single band at about 66 KDa was present. Thus,the Western blot studies confirm the immunoEM results that theCCK-B/gastrin-receptor is present in adenocarcinoma tumor cells,although the immunoEM studies do not distinguish between the isoforms ofthe CCK-B/gastrin-receptor. The data indicate that the presentimmunogens elicit anti-CCK-B/gastrin-receptor antibodies which canrecognize and bind various isoforms of the receptor, which would beadvantageous for the treatment of these tumors.

EXAMPLE 7

[0091] C170HM2 adenocarcinoma cells were injected intraperitoneally intonude mice and tumors were allowed to grow in the liver. Control micereceived an infusion of phosphate buffer saline solution (PBS) andexperimental mice received an infusion of oneanti-CCK-B/gastrin-receptor antibodies. In Group 1, each mouse wasinfused daily with 0.5 mg of Rabbit anti-CCK-B/gastrin-receptorantibodies generated against Peptide 1 (Rabbit anti-Peptide 1,Rbt@GRP1). In Group 2, each mouse received daily 0.5 mg of Rabbitanti-CCK-B/gastrin-receptor antibodies generated against Peptide 4(Rabbit anti-Peptide 4, Rbt@GRP4). The mice were studied for a period of40 days after antibody infusion, sacrificed and the tumors removed forstudy. The weight, size and cross-sectional area of the tumors wereassessed by standard techniques. The results are shown in FIGS. 9 and10.

[0092] As seen in FIGS. 9 and 10, implantation of the colorectaladenocarcinoma cancer cell line C170HM2 in mice without treatmentresults in the rapid growth of large tumor masses, as determined bytumor weight, or tumor size, and the tumor cross-sectional area of thetumors. However, infusion of the animals with Rabbit anti-Peptide 1 orRabbit anti-Peptide 4 antibodies results in a marked decrease in thenumber of animals having any detectable tumor, as well as in the weightand size of tumors in animals having them when compared to control. Thesame effect can be seen when mean tumor weight, mean tumor size, or meantumor number is calculated. These data are shown in FIGS. 11, 12 and 13.

[0093] Further insight into the distribution within the population isgained by calculating the medians of tumor numbers, weight and size. Theresults are shown in FIGS. 14, 15 and 16. As seen in these figures, theRabbit anti-Peptide 1 immunogen was consistently more effective thanRabbit anti-Peptide 4 in inhibiting tumor growth. However, both Rabbitanti-Peptide 1 and Rabbit anti-Peptide 4 antibodies did exhibit powerfultumor inhibitory activity as compared to the control treatment.

EXAMPLE 8

[0094] A larger tumor burden was generated in nude mice using the coloncancer cell line C170HM2 by a method as described in Example 7, but witha higher initial cell innoculum. The C170HM2 is a liver-invasivexenograft model. Control and experimental mice were treated also asdescribed in Example 7.

[0095] Forty days after antibody infusion, the mice were sacrificed andliver tumors were removed and studied. FIGS. 17, 18 and 19 show theresults of these experiments. FIG. 17 shows the mean and median livertumor numbers of control and anti-CCK-B/gastrin-receptor antibodytreated animals. The data show that theRabbit-anti-CCK-B/gastrin-receptor antibodies (“Rabbit@GRP”) areeffective in inhibiting the growth of the metastatic tumors in theliver. There is a statistically significant decrease in mean liver tumornumbers in mice livers using Rabbit anti- Peptide 1 (Student's T test),p=0.0084 and in the median liver tumor number, p=0.0016 (Mann Whitney)when compared to controls. Mice treated with anti-Peptide 4 antibodiesalso show a decrease in mean liver tumor number; however, there was nodifference in the mean liver tumor number in this animals when comparedto controls.

[0096]FIG. 18 shows that anti-Peptide 1 and anti-Peptide 4 antibodieswere also capable of reducing the mean and median tumor weights of livermetastases when compared to control animals. The data in FIG. 19 showthat anti-CCK-B/gastrin-receptor treated mice also had a significantdecrease in mean and median cross-sectional area of the liver tumorswhen compared to control animals.

[0097] The data indicate that the anti-CCK-B/gastrin-receptor antibodiesare effective in controlling the spread and growth of agastrin-dependent colon cancer in the liver, which constitutes the majorsite of metastatic spread of this cancer.

EXAMPLE 9

[0098] These studies we carried out to confirm GRP1 immunoreactivity onC170HM2 cells. The aim of the study was to evaluate tumor localizationof antiserum raised against GRP1 and to determine its therapeutic effecton the growth of C170HM2 cells within the liver of nude mice. C170HM2cells were injected intraperitoneally into nude mice as described inExamples 7 above. GRP1 antiserum was raised in rabbits. The antiserumwas radiolabelled with ¹²⁵I and administered to nude mice withestablished C170HM2 xenografts by a tail vein injection. Control micereceived ¹²⁵I radiolabelled normal rabbit serum. Mice were terminated atincreasing time points following injection of a single dose of ¹²⁵Iantibodies. Radioactivity was measured as counts per minute per gram of(CPM/g) tissue and the liver/liver tumor ratio calculated.

[0099]FIG. 20 is a graph which shows the radiolabeled rabbit anti-GRP1antibodies bound to liver tumors versus control. As seen in the figure,more rabbit anti-GRP1 antibodies are bound to liver tumor tissue whencompared to controls. FIG. 20 also shows the liver tumor/liver ratio onthe y axis with increasing time on the x axis for both radiolabelednormal rabbit serum and GRP1 antiserum. The normal rabbit serum achieveda ratio of 1 from day 1 which remained constant until day 5. Thisindicates the level of radiolabel in the liver tumour and normal liverwas equal. The ratio for GRP1 antiserum accumulated exponentiallyapproaching 2 by day 5. This indicates radiolabeled GRP1 antiserumspecifically localizes within C170HM2 liver tumors.

EXAMPLE 10

[0100] Therapeutic Effect of GRP1 Antiserum on C170HM2 xenografts

[0101] The C170HM2 tumor xenografts were initiated by intraperitonealinjections of cells. Three different cell inocula were used to generate3 levels of tumor burden. The GRP1 antiserum was administered passivelyby tail vein injection daily from day 0. Therapy was terminated on day40.

[0102] Effect of GRP1 Antiserum on Tumor ‘Take Rate’

[0103] The initial parameter evaluated was mean tumor number within theliver which is shown in FIG. 21. The normal rabbit antiserum treatedcontrols are grouped in increasing cell inocula. As seen in FIG. 21, inthe control groups the mean tumor number per liver was between 1 and 3.In the GRP1 antiserum treated group the mean tumor number per liver wasless than 1 for all three cell inocula, which was significant for all 3experiments (one inoculum, n=18, p=0.003; 2 inocula, n=12, p=0.0001 and3 inocula, n=20, p=0.0068, Mann Whitney analysis).

[0104] Effect of GRP 1 Antiserum on Tumor Weight of Established Tumors

[0105]FIG. 22 shows the mean tumor weight for the normal rabbit serumtreated controls on the left panel for the 3 increasing cell innocula.The figure also shows the mean tumor weight of nude mice followingtreatment with GRP1 antiserum the mean liver weight was reduced by 60%with all 3 cell innocula, which was significant for all 3 experiments(one inoculum, p=0.0016; 2 innocula, p=0.0084, and 3 innocula, p=0.0001,Mann Whitney analysis).

[0106] GRP1 Immunoreactivity in C170HM2 xenografts as Determined byWestern Blotting

[0107] Extra-nuclear membrane proteins were prepared from C 170HM2xenografts from 2/3 experiments. These were analyzed by Western blottingusing the GRP1 antiserum. FIG. 23 is a photograph of the Western blotshowing in the normal rabbit serum-treated xenografts 2 immunoreactivebands were present at 74 and 50 kDa, with the former band showing thestrongest immunoreactivity. In the GRP1 antiserum treated xenografts,there are 2 immuno-reactive bands together with an intermediate band,not seen in the control xenografts or cells grown in vitro. A 50 kDaband shows the strongest immunoreactivity. This indicates that in theGRP1 antiserum treated xenografts a larger proportion of theCCKB/gastrin-receptors may be present as an internalized form.

[0108] Histological Analysis of C170HM2 xenografts

[0109]FIG. 24 shows a microscopic view of a C170HM2 xenograft invading aliver of a nude mouse. The tumor is generally composed of a necroticcenter with a viable leading edge which squashes the hepatocytes as itinvades the liver. The degree of apoptosis was measured in the viableleading edge of C170HM2 tumors by the Tunel method with positive cellsvisualized by in situ hybridization. FIG. 25 shows that apoptotic cellswere present in the viable tumor cells in the GRP1 antiserum-treatedxenografts, but not in the normal rabbit serum-treated tumors.

[0110] The data show that antiserum raised against the amino terminalepitope of the CCKB/gastrin-receptor selectively localizes withinliver-invasive C170HM2 tumors. Neutralization of the GRP1 epitopeinduced a significant effect on both tumor ‘take rate’ and gross tumorburden of tumors that did establish. This tumor-inhibitory effect may bedue to (a) a general cytostatic effect induced by blocking theCCKB/gastrin-receptor and/or (b) an indirect effect of targeting anantibody to the nucleus of the cell, possibly resulting in apoptosis.

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1 3 17 amino acids amino acid not relevant linear peptide NO NON-terminal 1 Lys Leu Asn Arg Ser Val Gln Gly Thr Gly Pro Gly Pro Gly AlaSe 1 5 10 15 Leu 15 amino acids amino acid not relevant linear peptideNO internal 2 Gly Pro Gly Ala His Arg Ala Leu Ser Gly Ala Pro Ile SerPhe 1 5 10 15 7 amino acids amino acid not relevant linear peptide 3 SerSer Pro Pro Pro Pro Cys 1 5

We claim:
 1. An immunogen comprising a peptide from theCCK-B/gastrin-receptor conjugated to an Immunogenic carrier.
 2. Theimmunogen of claim 1, wherein the peptide has the amino acid sequenceKLNRSVQGTGPGPGASL (SEQ ID NO.: 1 in the Sequence Listing) orGPGAHRALSGAPISF (SEQ ID NO.: 2 in the Sequence Listing).
 3. Theimmunogen of claim 1 or 2, further comprising a spacer peptide sequence.4. The immunogen of claim 3, wherein the spacer peptide sequence isSSPPPPC (SEQ ID NO.: 3 in the Sequence Listing).
 5. The immunogen ofclaim 1, wherein the immunogenic carrier is selected from the groupconsisting of Diphtheria toxoid, tetanus toxoid and bovine serumalbumin.
 6. A method for treating a malignant condition caused bygastrin-dependent malignant cell growth comprising administering to ananimal in need of such treatment an effective amount of ananti-CCK-B/gastrin-receptor immunogen.
 7. The method of claim 6, whereinthe immunogen comprises a peptide from the CCK-B/gastrin-receptor. 8.The method of claim 7, wherein the peptide has the amino acid sequenceKLNRSVQGTGPGPGASL (SEQ ID NO.: 1 in the Sequence Listing) orGPGAHRALSGAPISF (SEQ ID NO.: 2 in the Sequence Listing).
 9. The methodof claim 8, wherein the immunogen further comprises a spacer peptidesequence.
 10. The method of claim 9, wherein the spacer peptide sequenceis SSPPPPC (SEQ ID NO.:3 in the Sequence Listing).
 11. The method ofclaim 10, further comprising an immunogenic carrier selected from thegroup consisting of Diptheria toxoid, tetanus toxoid and bovine serumalbumin.
 12. A method for treating a gastrin-dependent tumor comprisingadministering to an animal in need of such treatment an effective amountof anti-CCK-B/gastrin antibodies which recognize and bind to theCCK-B/gastrin-receptors in the tumor cells.
 13. The method of claim 12,wherein the antibodies are selected from the group consisting ofchimeric, monoclonal and humanized antibodies.
 14. The method of claim12, wherein the anti-CCK-B/gastrin-receptor antibodies recognize andbind the amino acid sequence KLNRSVQGTGPGPGASL (SEQ ID NO.: 1 in theSequence Listing) or GPGAHRALSGAPISF (SEQ ID NO.: 2 in the SequenceListing) of the receptor.
 15. The method of claim 12 or 14, wherein theantibodies are further conjugated to a cytotoxic molecule.
 16. Themethod of claim 15, wherein the cytotoxic molecule is a toxin or aradioactive molecule.
 17. The method of claim 16, wherein the toxin ischolera toxin.
 18. The method of claim 16, wherein the radioactivemolecule is labeled with ¹²⁵I or ¹³¹ I.
 19. A method for detecting agastrin-responsive tumor containing CCK-B/gastrin-receptors, comprisingexposing an anti-gastrin-receptor antibody to cells isolated from atumor biopsy sample and detecting the CCK-B/gastrin-receptor in thesample.
 20. The method of claim 19, wherein the anti-gastrin-receptorantibody is specific for an amino-terminal peptide of theCCK-B/gastrin-receptor.
 21. The method of claim 20, wherein the peptideis in the amino-terminal region of the CCK-B/gastrin-receptor comprisesamino acid residues 5-21.
 22. An immunogenic composition comprising ananti-CCK-B/gastrin-receptor immunogen.
 23. The composition of claim 22,wherein the immunogen comprises a peptide from theCCK-B/gastrin-receptor.
 24. The compostion of claim 22, wherein theimmunogen comprises antibodies against the CCK-B/gastrin-receptor.
 25. Amethod for diagnosing a gastrin-dependent tumor, comprisingadministering radiolabeled anti-CCK-B/gastrin-receptor antibodies to apatient possessing a colorectal tumor and imaging the tumor byscintigraphic scanning.
 26. The method of claim 25, wherein theanti-CCK-B/gastrin-receptor antibodies are radiolabeled with ¹¹¹Indiumor ⁹⁰Yttrium.